Rakash Nayar

Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34+ cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r=0.99), with no statistically significant difference or bias between methods (P > 0.05). Linearity of the absolute counting method generated an R2 value of 1.00 over the range of 0-250/microl. Precision of the absolute counting method measured at three concentrations of CD34+-stabilised KG1 a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34+ cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34+ cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibility of CD34 determinations due to the reduction in sample handling and calculation of results.

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.